Quantitative nucleic sequence analysis plays an increasingly important role in the fields of biological and medical research. For example, quantitative gene analysis has been used to determine the genome quantity of a particular gene, as in the case of the human HER-2 oncogene which is found at amplified levels in approximately 30% of human breast cancers. D. J. Slamon et al., Science 235, 177-182 (1987). More recently, gene and genome quantitation have also been used in determining and monitoring the levels of human immunodeficiency virus (HIV) in patients throughout the different phases of HIV infection and disease. M. R. Furtado et al., J. Virol. 69, 2092-2100 (1995). It has been suggested that higher levels of circulating HIV and failure to effectively control virus replication after infection may be associated with a negative disease prognosis; in other words, there may be an association between virus level (HIV replication) and the pathogenesis of the disease. M. Paitak et al., Science 259, 1749-1754 (1993). Accordingly, an accurate determination of HIV nucleic acid levels early in an infection may serve as a useful tool in diagnosing illness, while the ability to correctly monitor changing levels of viral nucleic acid throughout the course of an illness may provide clinicians with critical information regarding the effectiveness of treatment and progression of disease. Additionally, the determination of virion-associated HIV RNA levels in plasma represents a marker of viral replication with potential widespread applicability in assessment of the activity of antiretroviral therapy. ld.
Several methods have been described for the quantitative analysis of nucleic acid sequences. The polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) have permitted the analysis of small starting quantities of nucleic acid (e.g., as little as one cell equivalent). See, e.g., S. Edmands et al. 1994, PCR Methods Applic. 3, 317-19; I. R. Rodriguez et al. 1992, Nucleic Acids Res. 20, 3528. Early reports of quantitative PCR report quantitation of the PCR product, but do not directly measure the initial target sequence quantity. F. Ferre 1992, PCR Methods Applic. 2, 1-9. In general, these methods involve measuring PCR product at the end of nonisothermal amplification and relating this endpoint measurement level back to the starting nucleic acid concentration. Unfortunately, the absolute amount of product generated does not always bear a consistent and easily quantifiable relationship to the amount of target sequence present at the initiation of the amplification reaction. The kinetics and efficiency of amplification of a target nucleic acid sequence may also be strongly dependent on the starting abundance of the target sequence and the sequence match of the primers and target template. Thus, some RT-PCR amplification methods which rely on "endpoint" analysis may be capable of only revealing the presence or absence of the target nucleic acid sequence, but not the actual starting quantity with any degree of accuracy. For these reasons, comparison of the amount of specimen-derived PCR product to the amount of product from a separately amplified external control standard typically does not provide a highly accurate basis for quantitation.
One specific approach to nucleic acid amplification using PCR measures product quantity in the log phase of the reaction prior to the plateau. See, e.g., Kellogg et al. 1990, Anal. Biochem. 189, 202-208; S. Pang et al. 1990, Nature 343, 85-89. This method requires that each sample have equal input amounts of nucleic acid sequence and that each sample under analysis amplifies with identical efficiency up to the point of quantitative analysis. A gene sequence (contained in all samples at a relatively constant quantity) can be used for sample amplification efficiency normalization. However, using conventional methods of PCR detection and quantitation, it may be extremely laborious to assure that all samples are analyzed during the log phase of the reaction, both for the target gene and the normalization gene.
Another method referred to as quantitative competitive PCR (QC-PCR) has also been developed and is now widely used for PCR quantitation. See, e.g., P. D. Siebert and J. W Larrick 1992, Nature 359, 557-558; and X. Tan et al. 1994, Biochim. Biophys. Acta 1215, 157-162. QC-PCR relies on the inclusion of a known amount of an internal control competitor in each reaction mixture. The efficiency of each reaction is also normalized to the internal competitor. To obtain relative quantitation, the unknown target PCR product is compared with the known competitor PCR product, usually via gel electrophoresis. The relative amount of target-specific and competitor DNA is measured and used to calculate the starting number of target templates. Basically, in this kind of analysis, the larger the ratio of target specific product to competitor specific product, the higher the starting DNA concentration.
However, articles by Luc Raeymaekers, entitled "A Commentary on the Practical Applications of Competitive PCR", Genome Research 5, pp. 91-94 (1995) and "Quantitative PCR: Theoretical Considerations with Practical Implications" Analytical Biochemistry 214, pp. 582-585 (1993) (hereinafter, the "Raeymaekers 1995" and "Raeymaekers 1993" articles), suggest that PCR, by itself, may not be an accurate quantitation assay, notwithstanding its extreme sensitivity and specificity relative to other methods based on probe hybridization. In particular, because of the many amplification steps which take place during PCR, small differences in amplification efficiency may result in dramatic differences in product yield. Furthermore, because the exponential phase of the reaction is of limited duration (because of the accumulation of product), if PCR is run beyond the exponential phase into the saturation phase when endpoint analysis is performed, initial differences in the amount of template may become obscured. To compensate for some of these intrinsic difficulties associated with accurate quantitation using PCR, controls have been introduced. However, these controls may not adequately account for specific pitfalls associated with QC-PCR which uses an external standard sequence to facilitate quantitation.
As explained at page 92 of the Raeymaekers 1995 article, the prefix "QC" in QC-PCR refers to the fact that competition occurs between target and standard templates for available substrates when PCR is allowed to proceed into the saturation phase. Because the sum of the masses of both products cannot exceed some maximum value, the amount of product formed from one template will decrease with the increasing quantity of the other template. As will be understood by those skilled in the art, the products of target and standard sequences are discriminated either by a difference in length or by a specific restriction site in the region between the primer templates. In practice, a plurality of PCR tubes containing the same but unknown amount of target sequence is spiked with a dilution series of defined quantities of the standard. If the amplification factor is the same for both sequences, their ratio will remain constant during amplification and the amount of the unknown template can then be accurately quantitated from the ratio of the two products. Raeymaekers recommends that a "curve" be generated which relates the logarithm of the ratio of PCR products standard/target to the logarithm of the initial known amount of standard cDNA added (i.e., log (T.sub.n /S.sub.n) versus log (S.sub.0)). Here, Raeymaekers uses a plurality of samples and each sample has an aliquot portion of an unknown quantity of target and a respective known quantity of standard (S). The amount of initial target can then be read from the point on the curve where the amounts of target (T) and standard (S) are equal (i.e., where S/T=1 or log (S/T)=0).
Raeymakers also explains that if there is a difference in the amplification factor, theory predicts a parallel shift of the curve. This shift will cause a displacement in the point of equivalence and a faulty quantification because the magnitude of the displacement typically cannot be detected (because a reference point is typically not available). According to Raeymaekers, any determination that the curve has a slope of -1 does not suggest that the amplification factors are the same for both target and standard. From these considerations, Raeymaekers concludes that if a PCR assay yields a curve relating log (T.sub.n /S.sub.n) to log (S.sub.0) which is not linear or does not have a slope of -1 (or +1 in the event the abscissa provides log (T.sub.0)), it cannot be used for either absolute or relative quantitation. Moreover, the slope =-1 requirement for the curve is a necessary but not a sufficient condition for establishing that the amplification factors are the same for T and S and therefore not a sufficient condition for absolute quantitation. Instead, the requirement that the amplification factors are equal has to be independently demonstrated as a prerequisite to obtaining accurate absolute quantitation, without reliance on the curve, and such independent demonstration may be difficult to achieve particularly if the target and standard sequences are dissimilar. These conclusions are explained more fully at pages 584 and 92 of the Raeymaekers 1993 and 1995 articles, respectively.
Similar conclusions are also reached at page 632 of an article by G. Haberhausen et al., entitled "Comparative Study of Different Standardization Concepts in Quantitative competitive Reverse Transcription-PCR Assays" Journal of Clinical Microbiology, Vol. 36, No. 3, pp. 628-633 (1998). Finally, the attempts at quantitation which are illustrated by the curves of FIGS. 2, 4 and 6 of an article by M. Piatak et al., entitled "Quantitative Competitive Polymerase Chain Reaction for Accurate Quantitation of HIV DNA and RNA Species", BioTechniques, Vol. 14, No. 1, pp. 70-80 (1993), would appear to be flawed in view of Raeymaekers' conclusions and further because the absolute values of the slopes of these curves differ significantly from unity. The accuracy of the results of Piatak et al. which are predicted from "corrected" fluorescence indicia may also be limited because the indicia were not obtained in real-time during amplification, but only after termination of amplification.
Thus, notwithstanding these attempts to perform absolute quantitation using nonisothermal amplification techniques such as PCR, there continues to a be need for improved methods of accurately determining starting quantities of nucleic acid sequences undergoing amplification, which do not require the establishment of identical amplification factors as a prerequisite to performing absolute quantitation.